Method for analysis of large polymer molecules

ABSTRACT

Method of gel permeation chromatography, especially relating to the analysis of polymers. The method comprises introducing a sample of a dilute solution of the polymer into a chromatographic column and measuring the volume and concentration of the eluate. The sample volume must be of a size sufficient to produce a rise in concentration of polymer in the eluate of from zero to that of the sample. The molecular size distribution of the polymer may then be determined from the measurements taken.

nited States atet Moore [54] METHOD FOR ANALYSIS OF LARGE POLYMERMOLECULES [72] Inventor: John C. Moore, Lake Jackson, Tex.

[73] Assignee: The Dow Chemical Company, Midland,

Mich.

[221 Filed: Nov. 26, 1969 211 Appl. No.: 880,121

[52] U.S. Cl. 23/230 R, 23/230 A, 23/253 R, 23/253 A, 73/6L1C [51] Int.Cl ..G0ln 11/06, GOln 15/00 [58] Field otSearch..23/230,253;73/61.1,61.1 C

[56] References Cited OTHER PUBLICATIONS Snyder, Determination ofAsphalt Molecular Weight Dis- Mar. 14, 1972 tributions by Gel PermeationChromatography, Analytical Chemistry, Vol. 41, No. 10, August 1969.pages 1223- 1227.

Primary Examiner-Morris Or Wolk Assistant Examiner-R. E. SerwinAttorney-Griswold & Burdick, Raymond B. Ledlie and A. Cooper Ancona [57]ABSTRACT Method of gel permeation chromatography, especially relating tothe analysis of polymers. The method comprises introducing a sample of adilute solution of the polymer into a chromatographic column andmeasuring the volume and concentration of the eluate. The sample volumemust be of a size sufficient to produce a rise in concentration ofpolymer in the eluate of from zero to that of the sample. The molecularsize distribution of the polymer may then be determined from themeasurements taken.

6 Claims, 7 Drawing Figures PATENTEDMAR 14 1912 3,649,200

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Fit 2 x4 INVENTOR. John C. Moo e PAKNTEDMAR 1 I972 SHEET [1F 5 Fbt tEMVEQU hb wgvv QRMZOQQOm wmgfi n6 9953b ZQRDQM sa a jwxG INVENTOR. JohnC. Moore WMA HVTORNEYJ PAIENTEBMR 14 m2 SHEET '5 OF 5 INVENTOR. John 6'.M0 Ore wx whw HTTORNEYS METHOD FOR ANALYSIS OF LARGE POLYMER MOLECULESBACKGROUND OF THE INVENTION This invention relates to the separation oflarge polymer molecules in solution and more particularly relates to animproved method for the analytical separation of polymer samples bymeans of a chromatographic method employing porous materials.

The separation of larger polymer molecules in solution by means ofporous materials is now generally referred to as gel permeationchromatography (GPC). This technique has become widely accepted as amethod of analyzing polymers, as a method of separating polymers intofractions by molecular size and as an analytical tool in the productionof polymers. Unlike the older chromatographic techniques such asadsorption chromatography or ion exchange chromatography, gel permeationchromatography retards the smaller species more than the larger speciesof a given polymer series and therefore elutes the highest molecularweight materials first. Since both the results and the mechanism ofseparation are dissimilar to the adsorption and ion exchange techniques,some of the problems encountered are likewise dissimilar. Experiencewith the gel permeation separation process has shown that a relativelylong column is required to achieve good separation efficiency andtherefore 2 to 4 hours is usually required for the separation oranalysis of a polymer sample, especially when large molecular speciesare present. Attempts to shorten the column and consequently shorten theanalysis time have met with a disproportionate loss in accuracy.Separation efficiency varies sharply with load and, therefore, verydilute solutions of polymer have been employed since more concentratedsolutions of polymer tend to overload the column and thereby severelyimpair the separation.

Using well-characterized narrow fractions of a polymer as samples, acolumn may be calibrated in terms ofits separating and band-spreadingcharacteristics by known methods. A universal calibration" results whenmolecular size is expressed as hydrodynamic volume, that is, as theproduct of molecular weight and intrinsic viscosity. The molecularweight distribution of an unknown sample of a given polymer may then beobtained by finding that distribution which, when subjected to the knownseparating and band-spreading characteristics of the column, would havegiven the chromatogram. In all but the simplest cases this has beenaccomplished by iterative methods. For further discussion of the use ofthese methods refer to J. C. Moore, in Characterization ofMacromolecular Structure," Publication 1573, Nat. Acad. of Science,Washington, DC. 1968, pp. 273-284 and L. H. Tung, J. C. Moore and G. W.Knight, J. Appl. Polymer Sci. 10, 1261(1966).

It is preferred to have the heteroporous packing match the size range ofpolymer molecules being analyzed. Thus, the heteroporous packing isselected by referring to a calibration curve which indicates the rangeof molecular sizes separable by the given packing. If the range ofmolecular size of the polymer sample is unknown, the particular packingmaterial may be selected by a trial run of the sample through one ormore known packing materials.

It is to be understood that porous material of any range of sizes (i.e.,porosity) can be used so long as this range includes the range of poresizes which will make the desired separation.

Gel permeation chromatography columns have heretofore been operated bypassing a relatively small volume of dilute solution of a sample ofpolymer into a column packed with heteroporous material and filled withsolvent. After the sample has been injected into the column, additionalsolvent is injected to force the sample through the column wherebyseparation occurs. The solution then passes from the column to ananalytical means such as a differential refractometer.

In all forms of chromatography using small samples, as the sample iscarried through the column both the separation process and certain zonebroadening processes spread the concentration profile so that it becomeslower and broader. It has been observed in GPC that the small increasein sample viscosity due to the presence oflong chain molecules presentsan instability in the sample cross section at the trailing boundary ofthe sample. The more fluid solvent tends to flow more rapidly than therelatively viscous polymer solution through the small openings betweenthe particles of packing. This effect is called viscous fingering andcan occur throughout the sample zone where ever a less viscous fluiddisplaces a more viscous one. This process results in back mixing" andloss of efficiency. This effect is shown on the chromatogram as anirregular tailing of components and produces a larger decrease in columnefficiency under the main body of the eluted peak. This phenomenon isresponsible for the marked effect of sample concentration on theseparations shown in FIGS. 4 and 5, which will be more fully discussedlater. These effects seem to be peculiar to gel permeationchromatography separations and are relatively more serious as the lengthof the columns is reduced.

It is an object of this invention to provide an improved method for theanalysis of various high-molecular weight components of a polymer.

A further object of the invention is to provide a method which permits amore precise analysis of polymer components at increased polymerconcentrations in the solvent.

A still further object of the invention is to provide more rapidchromatographic analysis of polymers than has heretofore been possiblewithout losing efficiency.

These and other objects and advantages of the present invention willbecome apparent from the following detailed specification.

BRIEF DESCRIPTION OF THE INVENTION I have now discovered that when arelatively large sample of a dilute solution ofa polymer is introducedinto a GPC column filled with the same solvent used to dissolve thesample and flow is continued until all of that solvent is displaced bythe solution of sample, a transition zone occurs between solvent andsolution, in which zone the concentration of polymer molecules variesaccording to molecular size.

Thus, by using a sample large enough to produce a rise in polymerconcentration in the eluate of from zero to the concentration of thesample, a complete and more accurate analysis of the molecular sizedistribution in the sample can be obtained. The rising concentration ofpolymer in the eluate provides a condition in which the eluted solutionis continually increasing in viscosity and, therefore, viscous fingeringdoes not occur.

It has been found and will be demonstrated in the appended examples thatthe separation processes of GPC are at work in this transition zone andthat analysis of the data from this transition zone gives a molecularsize distribution of the sample.

Having found how to overcome the problem ofviscous fingering," apreviously unrecognized load effect was discovered. Mutual compressionof the long chain molecules, such that their effective size diminishesas their environmental concentration increases, results in a delay intheir elution beyond that point at which they are expected. This showsup especially when samples of two or more different average molecularweight polymers are used and in more highly concentrated solutions ofpolymers. In making an analysis by means of examining the transitionzone referred to above, which can also be referred to as frontalanalysis, the effect caused by mutual compression of the largemolecules, which will be called viscous delay," can be correlated withsolution properties and the chromatogram can be corrected by appropriatemathematics.

An expression which took into account a combination of molecularcompressibility and solution concentration enabled the prediction of thecompression for each molecular species. This compression factor wasapplied to compensate for the viscous delay.

DETAILED DESCRIPTION OF THE INVENTION In the practice of the presentinvention the effluent solution from the column is passed to an analyzersuch as a differential refractometer. The refractive index of thesolution relative to the pure solvent may be plotted to give a curvesuch as that shown in FIG. I. The data from the transition zone (between27 ml. and 35 ml.) may be converted by known methods to a differentialcurve such as that shown in FIG. 2 from which the molecular weightdistribution of the sample may be obtained.

Thus by employing frontal analysis techniques the problems of viscousfingering" and sample size are eliminated and the effect of viscousdelay" becomes predictable so that a compensating factor can be appliedto give a better analysis of the molecular size distribution in apolymer sample. Further, this analysis can be accomplished in a shorterperiod of time using solutions of normal concentration on short columns.

Since the method corrects problems which occur outside the pores of thecolumn packing, the invention is not limited to the use of anyparticular packing or eluting materials.

Suitable column packing for use in the process of this invention arenonpolar polymeric materials having a controlled and selectedheteroporosity such as are described in US. Pat. No. 3,326,875 and J.Polymer Sci. 62, 30l (I962). Other materials such as porous glass,silica, alumina, or any packing suitable for GPC as previously practicedmay be used. Porous polymers suitable for use as GPC column packings inseparating water soluble polymers in aqueous solutions have beendisclosed in a copending US. Pat. application Ser. No. 696,131, filedJan. 8, 1968, in which dextrans, polyethylenimines and the like areseparated on a heteroporous copolymer of acrylamide or methacrylamideand alkylidenebisacrylamide. The process of the present invention isalso applicable in separating these water soluble polymers.

It is expedient to have the heteroporous packing cover, or match, thesize range of the molecules being analyzed. This can be determined asknown to those skilled in the art.

Suitable eluting solvents including toluene, diethylbenzene, benzene,chloroform, perchloroethylene, tetrahydrofuran, dimethylformamide,trifluoroethanol and metacresol are use ful for conducting separationsof various organic polymers on the nonpolar porous polymeric gels; whilewater, aqueous buffer solutions, organic solventwater mixtures (e.g.,alcoholwater, acetone-water-chloroform), dimethyl formamide and dimethylsulfoxide are useful in conducting separations of water soluble typepolymers. In general a solvent is useful if it dissolves the polymer andmatches the polarity of the gel well enough, or differs from it in theright direction so that retention (delay) of the sample components doesnot occur by adsorption or preferential solvation, and the elution ofthe sample occurs in the order of decreasing molecular size. Theserequirements are well known to those skilled in the art of gelpermeation chromatography.

Substantially any polymer which is soluble in a useable solvent may beanalyzed by the process of this invention. Such polymers includepolyolefins such as polyethylene and polypropylene, polyglycols,polystyrene, polyvinyl chloride, polyesters, polyamides, alkyd resins,synthetic rubbers, cellulose ethers, cellulose esters, polyacrylic acidsalts, polyamines, and the like.

Where a recording differential refractometer is employed for analysis offractions eluted during the practice of this process, the elution curveis equivalent to the integral of the usual small sample elution curve(See FIG. 1). Such curve may then be differentiated graphically oranalytically (See FIG. 2A). In standard practice, effluent solution fromthe chromatographic column is passed through one side of thedifferential refractometer while a material (usually the solvent) havinga constant refractive index is passed through the other side. In apreferred embodiment of this invention, however, the differential curveis produced directly by the recording differential refractometer bypassing the eluate solution from the sample side of the cell through ashort capillary delay line and then through the reference side of thecell (FIG. 3).

The volume of the loop carrying the eluate from the sample side of therefractometer cell to the reference side depends upon the resolutiondesired to obtain the useful data available in the chromatogram and theneed to keep "noise" at a minimum. The loop volume is conveniently aboutI to 2 percent of the pore volume of the gel.

In the practice of the present invention a molecular size distributionof a polymer is obtained by l passing a sample ofa dilute solution ofthe polymer through a column containing heteroporous packing material,wherein the sample has a volume sufficient to give a rise in theconcentration of polymer in the eluate of from zero to that of thesample. (2) measuring this rise in concentration as a function ofelution volume, (3) differentiating this series of measurements toobtain the molecular size distribution of the polymer and (4) applying afactor to this distribution to correct for band spreading andconcentration (or load) effects to obtain a more accurate molecular sizedistribution of the polymer.

In applying this method to process control of a polymerization reactiona dilute solution of the reaction product, or of the reaction mediumduring the course of the reaction, can be passed through a column ofporous material (which matches the size of the polymer productmolecules) to obtain a frontal rise of concentration in the eluate up tothat of the sample. The concentration rise is measured as a function ofeluate volume and these data are compared to like data identicallyobtained from a standard sample of the desired product. From thisinformation the process parameters can be adjusted as necessary toobtain the desired product.

The measurement of concentration and volume can be taken in incrementsor the measurement can be made in a continuous manner as is the casewhen the eluate is conducted through a differential refractometer.

The following examples are representative of the method of theinvention.

EXAMPLE I In this experiment, a standard 48X% inch 0.!). column packedwith 3X10 A. permeability gel was employed. The column efficiency was1,900 plates per foot as determined with a very small benzene sample. Acontinuous differential refractometer was employed in combination with arecorder having a chart speed of 30 inches per hour to monitor theeluate concentration.

A sample of 25 ml. ofa solution containing 0.25 mg./ml. of a polystyrenehaving a average molecular weight of 41 1,000 in tetrahydrofuran wascontinuously introduced into the column at a temperature of 25 C. and aflow rate of 1 ml. per minute until the curve produced by the recorderbecame flat showing that full sample concentration had been reached. Thecurve thus produced, shown in FIG. I, was differentiated by thegraphical method to produce the curve shown in FIG. 2A which curve isequivalent to the normal elution curve of a small sample (0.8 ml.) ofthe same polystyrene shown in FIG. 2B.

In each of the following examples the conditions of temperature and flowrate were the same as in Example 1.

EXAMPLE 2 A modification of the refractometer was made by connecting thetwo sides of the cell with a 235 mm. length of one-sixteenth inchstainless steel tubing having an inside diameter of 0.037 in. and aninternal volume of 0.17 ml. By this means the effluent from the sampleside of the cell was passed through the reference side. In this mannerthe differential curve was produced directly. FIG. 3 shows the resultsobtained by this modification when using a sample of the same polymer asused in Example 1.

EXAMPLE 3 Viscous fingering and the effect of concentration are shown inFIG. 4. Using the same chromatographic column as in Example 1, threeseparate (0.8 ml.) samples of a polystyrene, having a weight averagemolecular weight of 1,800,000, in tetrahydrofuran were injected. Thefirst (lower curve) contained 0.0625 mg./ml. of polymer; the second(middle curve) contained 0.250 mg./ml. of polymer; and the third (uppercurve) contained 1,00 mg./ml. of polymer. Peak broadening is noticeablein the middle curve, but the peak occurs at essentially the same pointas in the lower curve, about 26.0 ml. The sample of highestconcentration, however, shows a distinct skewing ofthe curve and a shiftof the peak to the right at 26.8 ml., a pronounced tailing effect and abump in the curve occurring after the major peak.

Thus, viscous fingering is shown to cause errors in the chromatogram asconcentration increases. With larger samples the effect is still morepronounced.

EXAMPLE 4 A. Employing the method of Example 3, in which a small sample(0.8 ml.) was used. a mixture of equal weights of three differentpolystyrenes, having weight average molecular weights of 173,000, 411,000 and 1,800,000, respectively, was chromatogrammed. Three runs weremade in which the concentrations with respect to each component were0.0625, 0.25 and 1.00 mg/ml., respectively. That is, the first runcontained 0.0625 mg./ml. of each of the three polystyrenes, or a totalof 0.1875 mg./ml. of the polymers. The second and third runs similarlycontained a total of 0.75 and 3.0 mg./ml. of the polymers, respectively.The results, seen in H6. 5, show the errors introduced by viscousfingering as concentration is increased Note that the first peak,especially at the highest concentration (upper curve) is the mostseverely distorted. In fact, distortion is so great that a portion ofthe component molecules is eluted sufficiently late to obscure thesecond peak. This component (1,800,000 mw.) is the one excluded by thepores of the gel and, thus, shows that viscous fingering is aninterstitial phenomenon.

B. A large sample (25 ml.) of the same mixture used in A above waspassed through the same chromatographic column to obtain a frontalconcentration rise. This rise was differentiated by the recordingrefractometer to give the three curves shown in FIG. 6. Here, theviscous fingering effect has been eliminated as shown by the lack ofdistortion of the first (highest m.w.) peak, even at the highestconcentration. Another phenomenon associated with increasingconcentration, however, is apparent. This is the viscous delay" referredto earlier in the description sections which causes each species toelute from the column later then expected. Thus, in the case of anunknown, an erroneous reading is given as to its apparent molecularweight. Note that no delay occurs with the highest molecular weightcomponent, the first peak, since even though mutual compression of themolecules occurs, none is yet compressed sufficiently to enter the porestructure and be delayed. The progressive delay becomes apparent withthe second and third component peaks.

It has been found that the viscous delay effect can, at least as a firstapproximation, be formulated as a compression of molecular hydrodynamicvolume, proportional to the product of l) a mutual pressure or crowdingfactor, and (2) a compressibility factor for the particular molecularspecies whose viscous delay is being calculated. The crowding factor 1)may be taken as the specific viscosity of the solution in very dilutesolutions the sum of the products of the concentrations and theintrinsic viscosities of all the polymeric solutes. At present thiscompressibility must be experimentally determined for a given polymerand solvent. It has been observed that the molecular compressibilityCM.- shows an increase with where X and Y have values on the order of0.7 and 0.2, respectively, and [1; I, is the intrinsic viscosity ofthemolecular species 1' whose viscous delay is being sought.

In an iterative correction for band-spreading in the chromatogram, itmay be assumed for the first iteration that the viscous delay effect isnegligible, and a distribution of molecular weights and relativeconcentrations will be obtained. Since in frontal analysis the elutedconcentrations are cumulative, and the final concentration is that ofthe sample, the calculation may be caused to yield the cumulativespecific viscosity at each point in the frontal chromatogram.Multiplying these values by the compressibility of the species whosemean is assumed to be located at each point, a series of compressionfactors results. Applying these to the hydrodynamic volumes of theassumed species, a new set of hydrodynamic volumes results, those of thecompressed species which now appear later than their noload positions.The next iterative correction for band-spreading may then employ theintrinsic viscosities of the compressed species and their band-spreadingeffects for a new calculation of the distribution and the cumulativespecific viscosity in the frontal Chromatogram. After a few iterations asatisfactory agreement is obtained. Example 5 shows how such acalculation compensated satisfactorily for the viscous delay effect.

EXAMPLE 5 In order to show the effect of viscous delay, a series ofchromatograms was obtained by running samples of each of two differentmolecular weight polymers through a short column (one-half the length ofthe column in the preceding examples) at various concentrations, usingfirst small samples (1.0 ml.) and then large samples (25 ml.). Theformer produced normal elution curves in which viscous fingering effectsare apparent, while the latter produced a frontal rise which wasdifferentiated by the refractometer.

The chromatographic column employed for these experiments comprised twocolumns connected in series. The first was a stainless steel tube 7.8mm. ID. X 12 inches long packed with 10 A. permeability styrene-divinylbenzene copolymer gel. The second column of the same size was packedwith 10 A. permeability gel. These were connected with a short length ofstainless steel capillary tubing having a volume of 0.15 ml. When thefrontal analysis curve was differentiated by the refractometer, a loopconnecting the sample side with the reference side was used which had avolume of0.26 ml.

Calculated molecular weights are shown in the table below which, in thecase of the small samples, have been corrected for peak broadeningeffects, and in the case of the large samples, have additionally beencorrected for the viscous delay. The factor applied to correct forviscous delay is the product of the specific viscosity and thecompressibility factor indicated between Examples 4 and 5 above. Theapparent molecular weight of the sample is shown for each concentration(without correction) for comparison with the corresponding molecularweights which have been corrected for viscous delay and peak broadeningeffects. The ratios of weight to number average molecular weight alsoare given to show the deviation from the known ratio for the originalsample.

TABLE 1 Small Sample Chromatogram 1.0 ml.)

TABLE 11 Large Sample-Frontal rise-Chromatogram (25.0 ml.)

A Single run: all others are averages ofiwo runs The Mw./Mn. ratio forthe original samples of polystyrene were 1.20 for the 1,800,000 and 1.05for the 411,000 molecular weight samples.

Note the small change in apparent molecular weight and in the Mw./Mn.ratio for the low-molecular species as compared to that for thehigh-molecular species as concentration increases in the small sampleruns in Table I. The method of the present invention permits the moreaccurate determination of molecular weight distribution as shown by therelatively constant apparent molecular weight and Mw./Mn. ratio for bothhighand low-molecular species as shown in the corrected columns of Table11. The method of this invention, thus enables the use of samples ofhigher concentration and therefore better resolution of minor componentsin the analysis of high polymers.

Iclaim:

1. The method of analyzing a polymer for its molecular size distributionby means of gel permeation chromatography in which a dilute solution ofpolymer is passed through a porous packing material which comprises:

1. passing a sample of said polymer solution through said packing,wherein said sample has a volume sufficient to produce a rise inconcentration of polymer in the eluate of from zero to the concentrationof the sample,

2. measuring said rise in concentration as a function of the volume ofsaid eluate,

3. differentiating said measurement to obtain a molecular sizedistribution of said polymer and 4. applying a factor to saiddistribution to correct for band spreading and load effects.

2. The method of claim 1 wherein the measurement of concentration riseis obtained by a differential refractometer.

3. The method of claim .2 wherein the differentiation is obtained byconducting the eluate first through the sample side of the refractometerand then, by means of a short capillary delay line, through thereference side of the refractometer.

4. The method of claim 1 wherein the measurement of concentration riseis incremental.

5. The method of controlling a polymerization reaction by means of gelpermeation chromatography which comprises,

1. providing a column containing a porous packing material wherein thepores of said material match the size of the molecules of the polymerformed in said reaction,

2. passing a sample of a dilute solution of the product of said reactionthrough said porous packing material to obtain a frontal rise of eluateconcentration of polymer of from zero to the sample concentration,

3. detecting and measuring said frontal rise of concentration and 4.changing the reaction conditions to obtain the desired olymerization. 6.he method of claim 5 wherein the means of detecting and measuring thefrontal concentration rise is a differential refractometer.

2. The method of claim 1 wherein the measurement of concentration riseis obtained by a differential refractometer.
 2. passing a sample of adilute solution of the product of said reaction through said porouspacking material to obtain a frontal rise of eluate concentration ofpolymer of from zero to the sample concentration,
 2. measuring said risein concentration as a function of the volume of said eluate, 3.differentiating said measurement to obtain a molecular size distributionof said polymer and
 3. detecting and measuring said frontal rise ofconcentration and
 3. The method of claim 2 wherein the differentiationis obtained by conducting the eluate first through the sample side ofthe refractometer and then, by means of a short capillary delay line,through the reference side of the refractometer.
 4. The method of claim1 wherein the measurement of concentration rise is incremental. 4.changing the reaction conditions to obtain the desired polymerization.4. applying a factor to said distribution to correct for band spreadingand load effects.
 5. The method of controlling a polymerization reactionby means of gel permeation chromatography which comprises,
 6. The methodof claim 5 wherein the means of detecting and measuring the frontalconcentration rise is a differential refractometer.